Process for producing sustained-release preparation

ABSTRACT

The present invention is to provide sustained-release microcapsules which contains high amount of a drug, suppresses initial release and shows stable release, and the production method of which comprises adding a physiologically active substance to biodegradable polymer in an organic solvent containing a fat and oil (in particular, vitamin E) and dispersing and emulsifying the mixture.

FIELD OF THE INVENTION

[0001] The present invention relates to a sustained-release microcapsulewhich suppresses initial release of an excess amount of aphysiologically active substance right after administration of themicrocapsule and releases stably a constant amount of thephysiologically active substance for a long time from right afteradministration of the microcapsule, and a production method thereof.

BACKGROUND OF THE INVENTION

[0002] On sustained-release microcapsules of various physiologicallyactive polypeptides or low molecular water-soluble drugs, many reportshave been made [Critical Reviews in Therapeutic Drug Carrier Systems,12, 1-9 (1995); JP-A H2(1990)2-503315; EP-A-0586238; J. Pharm. Sci., 75,750-755 (1986); JP-A S57(1987)-118512]. Most of the microcapsules so farreported have the following drawbacks:

[0003] (1) in the manufacturing process, the amount of the water-solubledrug leaked to the outer aqueous phase is relatively large to invite arelatively low entrapment ratio of the drug, (2) the resultingmicrocapsules are generally porous and cause a relatively large initialrelease, and (3) in the manufacturing process, the physiologicallyactive substance is denatured to invite insufficient bioavailability.Thus, at the present stage, sustained release of the drug over adesirable long period have not yet been succeeded.

[0004] In JP-A S61(1986)-63613, improvement of sustained release ofmicrospheres was reported. That is, there is described that for thepurpose of preventing decrease of release rate of the active ingredienta certain hour after administration of microspheres whose base ispolylactic acid, in an organic solvent of polylactic acid to which theactive ingredient is dispersed, an oil soluble additive (medium chainfatty acid triglyceride, a lower fatty acid triglyceride, etc.) which issoluble in said solvent and which is biodegradable is uniformlydissolved. However, there is no suggestion on application to the otherbases nor on preparation of microcapsules using a solution of the activeingredient.

[0005] In JP-A H8(1996)-151321 [EP-A-0709085], there is disclosedmicrocapsules which contains an amorphous type water-solublephysiologically active substance and polymer, and which are producedfrom a S/O/W type emulsion. However, there is no description on aprocess for producing microcapsules using a solution of a drug as aninner aqueous phase nor on a method using metal complex of awater-soluble physiologically active peptide.

[0006] In EP 0765660, there is disclosed microcapsules which contains anamorphous type 2-piperazinone-1-acetic acid derivative, and in aproduction method thereof, a S/O/W type emulsion is employed. However,there is no description on a process for producing microcapsules using asolution of a drug as an inner aqueous phase nor on a method using metalcomplex of a water-soluble physiologically active peptide.

[0007] In general, in a process for producing microcapsules of awater-soluble physiologically active substance, it is more advantageousto employ a W/O type emulsion than a S/O type emulsion where the drug isused as solid substances in view of equivalency of drug content oroperation, and it is desired to employ a W/O type emulsion in anindustrial manufacture with large scale.

OBJECT OF THE INVENTION

[0008] It is preferable for sustained-release preparations usingbiodegradable polymers to suppress initial release of an excess amountof a physiologically active substance, in particular release of anexcess amount of the same within one day after administration thereof,and to releases stably a constant amount of the physiologically activesubstance for a long time. The present invention is to provide a simpleand convenient process for producing uniform sustained-releasemicrocapsules which maintain physiological activity of thephysiologically active substance, suppress initial release, and releasestably a constant amount of the physiologically active substance.

SUMMARY OF THE INVENTION

[0009] The present invention have intensively studied to solve the aboveproblems and, as a result, have found that in a process for producingsustained-release microcapsules of a water-soluble physiologicallyactive substance, it is possible to produce very usefulsustained-release microcapsules which suppress initial release of anexcess amount of the physiologically active substance right afteradministration and release stably a constant amount of thephysiologically active substance for a long time, by adding about 3% toabout 30% of a fat and oil to an organic solvent solution of saidbiodegradable polymer and using the thus obtained uniform solution as anoil phase. Further diligent studies based on this finding have reachedthe accomplishment of the present invention.

[0010] The present invention relates to

[0011] (1) A process for producing a sustained-release microcapsule of awater-soluble physiologically active substance, which comprises forminga w/o type emulsion comprising a solution containing a water-solublephysiologically active substance as an inner aqueous phase and anuniform organic solvent solution containing (i) a biodegradable polymerand (ii) a “fat and oil” as an oil phase, and removing the organicsolvent;

[0012] (2) A process as described in the above (1), wherein the w/o typeemulsion is dispersed in an aqueous phase, and the organic solvent isremoved by in-water drying;

[0013] (3) A process as described in the above (1), wherein the inneraqueous phase is a solution containing a water-soluble physiologicallyactive substance and a basic substance;

[0014] (4) A process as described in the above (1), wherein thewater-soluble physiologically active substance is a polypeptide themolecular weight of which ranges from about 200 to about 80,000;

[0015] (5) A process as described in the above (1), wherein thewater-soluble physiologically active substance is an integrinantagonist;

[0016] (6) A process as described in the above (5), wherein the integrinantagonist is a GPIIb/IIIa antagonist;

[0017] (7) A process as described in the above (6), wherein theGPIIb/IIIa antagonist is a 2-piperazinone-1-acetic acid derivativerepresented by the formula (I):

[0018] wherein A¹ and A² are independently a proton-accepting group or agroup convertible into a proton-accepting group, D is a spacer having a2- to 6-atomic chain optionally bonded through a hetero atom and/or a 5-or 6-membered ring (provided that the 5- or 6-membered ring is countedas 2- or 3-atomic chain, depending on its bonding position), R¹ is ahydrogen atom or hydrocarbon group, R² is a hydrogen atom or a residualgroup formed by removing —CH(NH₂)COOH from an α-amino acid, or R¹ and R²may be combined to form a 5- or 6-membered ring, P is a spacer having a1- to 10-atomic chain optionally bonded through a hetero atom and/or a5- or 6-membered ring (provided that the 5- or 6-membered ring iscounted as 2- or 3-atomic chain, depending on its bonding position), Yis an optionally esterified or amidated carboxyl group, and n is aninteger of 0-8; or a salt thereof;

[0019] (8) A process as described in the above (7), wherein the2-piperazinone-1-acetic acid derivative (I) is(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid or a salt thereof;

[0020] (9) A process as described in the above (7), wherein the2-piperazinone-1-acetic acid derivative (I) is(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid hydrochloride;

[0021] (10) A process as described in the above (7), wherein the2-piperazinone-1-acetic acid derivative (I) is(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticaciddihydrochloride;

[0022] (11) A process as described in the above (1), wherein thebiodegradable polymer is an aliphatic polyester;

[0023] (12) A process as described in the above (11), wherein thealiphatic polyester is a lactic acid/glycolic acid copolymer;

[0024] (13) A process as described in the above (1), wherein the fat andoil is an oil soluble vitamin;

[0025] (14) A process as described in the above (13), wherein the oilsoluble vitamin is α-tocopherol;

[0026] (15) A process as described in the above (1), wherein the finalconcentration of the fat and oil in a whole content of thesustained-release microcapsule is about 3% to about 30% (w/w);

[0027] (16) A process as described in the above (1), wherein the W/Otype emulsion comprising a solution containing a water-solublephysiologically active substance and a basic substance as an inneraqueous phase and an uniform organic solvent solution containing abiodegradable polymer and a fat and oil as an oil phase is dispersed inan aqueous phase to form a W/O/W type emulsion, and the organic solventis removed by in-water drying;

[0028] (17) A process as described in the above (3) or (16), wherein thebasic substance is a basic amino acid;

[0029] (18) A process as described in the above (17), wherein the basicamino acid is L-arginine;

[0030] (19) A process as described in the above (3) or (16), wherein thefinal concentration of the basic substance in a whole content of thesustained-release microcapsule is about 1% to about 8% (w/w);

[0031] (20) A process for producing a sustained-release microcapsule,which comprises removing an organic solvent from a S/O type dispersionwhere a metal complex of a water-soluble physiologically active peptideis dispersed in an uniform organic solvent solution containing abiodegradable polymer and a fat and oil;

[0032] (21) A process as described in the above (20), wherein the S/Otype dispersion is dispersed in an aqueous phase to form a S/O/W typeemulsion, and the organic solvent is removed by in-water drying;

[0033] (22) A process as described in the above (20), wherein thewater-soluble physiologically active peptide is human growth hormone;

[0034] (23) A process as described in the above (20), wherein the metalcomplex of the water-soluble physiologically active peptide is a zinccomplex of human growth hormone;

[0035] (24) A sustained-release microcapsule produced by the processaccording to the above (1);

[0036] (25) A sustained-release microcapsule produced by the processaccording to the above (20);

[0037] (26) Use of a fat and oil for the manufacture of asustained-release microcapsule of a water-soluble physiologically activesubstance, said microcapsule being produced by forming a w/o typeemulsion comprising a solution containing the water-solublephysiologically active substance as an inner aqueous phase and anorganic solvent solution containing a biodegradable polymer as an oilphase, and removing the organic solvent;

[0038] (27) Use of a fat and oil for the manufacture of asustained-release microcapsule of a metal complex of a water-solublephysiologically active peptide; etc.

BRIEF DESCRIPTION OF THE DRAWING

[0039]FIG. 1 shows the time-course changes of the plasma level of thedrug after administration of the microcapsules used in Test Example 1.

[0040]FIG. 2 shows the time-course changes of the plasma level of thedrug after administration of the microcapsules used in Test Example 2.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The abbreviations of amino acid, peptide, protecting group, etc.in this specification are based on those established by IUPAC-IUBCommission on Biochemical Nomenclature or those commonly used in therelevant fields. When optical isomers of amino acids are present, theyare L-isomers unless otherwise specified. The term “microcapsule” usedin this specification includes microspheres, microcapsules,microparticles, nanoparticles, nanospheres, nanocapsules, etc. whichcontain a physiologically active substance and a polymer.

[0042] The term “S/O/W type emulsion” and “W/O/W type emulsion” used inthis specification means a solid/oil/water (solid phase in oil in water)type emulsion and a water/oil/water (water phase in oil in water) typeemulsion, respectively.

[0043] Examples of the water-soluble physiologically active substancesinclude any water-soluble drugs such as a physiologically activepolypeptide; a compound used as anti-platelet aggregation agents (e.g.integrin antagonist), anti-tumor agents, antibiotics, antipyretics,analgesics, anti-inflammatory agents, antitussive expectorants,sedatives, muscle relaxants, antiepileptic agents, antiulcer agents,antidepressants, antiallergic agents, cardiotonics, antiarrhythmicagents, vasodilators, hypotensive agents, diuretics, antidiabeticagents, anticoagulants, hemostatics, antituberculous agents, hormonepreparations, narcotic antagonists, bone resorption inhibitors,osteogenesis promoting agents, angiogenesis inhibitors, etc.; etc.

[0044] Examples of said water-soluble physiologically active substancesinclude physiologically active substances whose water-solubility at 25°C. is not less than 0.1% (w/w), preferably not less than 1% (w/w).

[0045] The physiologically active polypeptide as a component of thepresent invention is exemplified by various peptides or proteins thatpossess physiologically activity beneficial to mammals and that can beused clinically. Said “physiologically active polypeptide” has amolecular weight of, for example, about 200 to about 200,000, calculatedon a monomer basis, preferably about 200 to about 80,000. Preferredphysiologically active polypeptides include polymers classified in thebiological field as proteins having higher structure. Any kind ofphysiologically active polypeptides can be used for the presentinvention, as long as the object of the present invention isaccomplished. Typical examples include growth factors, cytokines,enzymes, hormones, etc. More specifically, the following peptides andprotein may be mentioned as examples:

[0046] (1) Examples of the growth factors include nerve growth factor(NGF-1, NGF-1, etc.), nerve trophic factor (NTF), epidermal growthfactor (EGF), platelet-derived growth factor (PDGF), insulin-like growthfactor (IGF-1, IGF-2, IGF-3, etc.), fibroblast growth factor (aFGF,bFGF), osteogen growth factor (BMP-1, BMP-2, BMP-3, BMP-4, etc.), atrialnatriuretic factor (ANP), cartilage induction factor, etc.

[0047] (2) Examples of the cytokines include interferon (IFN-α, IFN-β,IFN-γ, etc.), interleukin (IL-1 to IL-11, etc.), cachectin, oncostatin,colony-stimulating factor (G-CSF, M-CSF, GM-CSF, etc.), trombopoietin(TPO), erythropoietin (EPO), etc.

[0048] (3) Examples of the enzymes include tissue plasminogen activator(tPA), urokinase (UK), streptokinase, protein C, metalloprotease,superoxide disumutase (SOD), Factor VIII and IX, etc.

[0049] (4) Examples of the hormones include growth hormone (GH), growthhormone-releasing factor (GRF), insulin, glucagon, gastrin, prolactin,adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone (TSH),follicle-stimulating hormone (FSH), luteinizing hormone (LH), humanchorionic gonadotropin (HCG), calcitonin, etc.

[0050] Preferable examples of said physiologically active polypeptideinclude hormones [e.g. growth hormone (human growth hormone, etc.),insulin (human insulin, etc.), etc.], cytokines (e.g. interferon,interleukin, etc.), etc.

[0051] The physiologically active polypeptide for the present inventioninclude polypeptides naturally derived and synthesized, semi-synthesizedor recombinant polypeptides produced by gene recombination or peptidesynthesis [e.g. recombinant human growth hormone (hereinafter, referredto as rhGH)]. Such polypeptide may have a sugar chain, and the structureof said sugar chain may be different from that of natural polypeptide.Also, they include muteins, derivatives, analogues, active fragments,etc. of the physiologically active polypeptide or protein. Hereinafter,the terms “physiologically active polypeptide”, “growth hormones”,“insulins”, “interferons” and “interleukins” are to be understood toinclude respectively those having a sugar chain and their muteins,derivatives, analogues and active fragments. When the physiologicallyactive polypeptide is muteins, derivatives or analogues of an optionalpolypeptide, its mechanism of action may be either agonistic orantagonistic.

[0052] The physiologically active polypeptide for the present inventionmay be in a form of complex with a metal atom. Examples of the metalcomplex of the polypeptide include a water insoluble (or hardly soluble)polyvalent metal complex, metal salt, etc. of the polypeptide. Any metalmay be used for the metal complex without limitation, as long as it is ametal that does not adversely affect the living body. For example, awater-soluble polyvalent metal (divalent, trivalent or tetravalentmetal, e.g. transition metal such as iron, copper, zinc, etc., IIIbgroup metal such as aluminum, etc., Ivb group metal such as tin, etc.)is preferably used.

[0053] Examples of the metal complex of the polypeptides includephysiologically active polypeptide metal salts obtained by contacting aphysiologically active polypeptide with a water-soluble polyvalent metalsalt (e.g. a salt of the above polyvalent metal with an inorganic acidsuch as hydrochloric acid, sulfuric acid, nitric acid, thiocyanic acid,etc. or a salt of the above polyvalent metal with an organic acid suchas aliphatic carboxylic acid (e.g. aliphatic mono-, di- ortri-carboxylic acid, preferably, aliphatic carboxylic acid having 2-9carbon atoms, etc.), aromatic acid (e.g. benzoic acid, salicylic acid,etc.), etc.). Said physiologically active peptide metal salt can beproduced by mixing a physiologically active polypeptide with awater-soluble polyvalent metal salt in a solution whose pH is selectedfrom the scope where solubility of both the reactants is not extremelydecreased.

[0054] Examples of ratio (mole ratio) of the physiologically activepolypeptide and the water-soluble polyvalent metal salt include 1:1 to1:1000, preferably 1:1 to 1:100, more preferably 1:1 to 1:50. It ispreferable to employ concentration of both of the reactants which iswithin the range of solubility of respective reactant and which is overthe range of solubility of the resulting complex. If necessary, thesolution to be employed may be adjusted to a weakly acidic, neutral orweakly basic solution.

[0055] When the physiologically active polypeptide have an acidic group(e.g. carboxyl group, sulfo group, etc.), it is advantageous to use itswater-insoluble complex (or complex which is hardly soluble in water)with a polyvalent metal for the purpose of improvement of entrapmentinto microcapsule and control of release.

[0056] Examples of the anti-platelet aggregation agents include integrinantagonists, in particular, GPIIb/IIIa antagonists. Examples of theGPIIb/IIIa antagonists include snake venom peptide (e.g. barbourin, orpeptides having amino acid sequence: Arg-Gly-Asp such asArg-Gly-Asp-Ser, (Arg-Gly-Asp-Ser) tetramer, Gly-Arg-Gly-Asp-Ser-Pro,cyclo-S,S-[AC-Cys(N α-methyl)Arg-Gly-D-Asn-penicillamine]-NH₂(SK&F-106760: Pharm. Res., 11, 1358-1390, 1994), and a compound havingan activity similar to GPIIb/IIIa antagonistic activity such as(S)-4-[(4-amidinobenzoyl)glycyl]-3-methoxy-carbonylmethyl-2-oxopiperazine-1-aceticacid, 4-(4-amidinobenzoylglycyl)-2-oxopiperazine-1,3-2-acetic acidhydrochloride,L-Tyr-N-(butylsulfonyl)-0-[4-(4-piperidinyl)butyl]monohydrochloride(L-700462/MK-383: Circulation, 88, 1512-1517, 1993), ethyl3S-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino-4-pentinoatehydrochloride (SC-54684A: Circulation, 91, 403-410, 1995),[1-[N-(P-amidinophenyl)-L-Tyr]-4-piperidinyl]acetic acid (Ro-44-9883:Thromb. Haemostas., 70, 817-821, 1993),cyclic[D-2-aminobutyryl-N-2-methyl-L-Arg-Gly-L-Asp-3-aminomethyl-benzoicacid]methane sulfonate (DMP728: Circulation, 89, 3-12, 1994),(3S-trans)-5-[[[4′-(aminoiminomethyl)-[1,1′-biphenyl]-4-yl]oxy]-methyl]-2-oxo-pyrrolidine-3-aceticacid (Fradafiban; BIBU 52: Circulation, 96, 1130-1138, 1997) representedby the formula:

[0057]2(S)-[(p-toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4-yl)ethyl]-4H-pyrazoro[1,5-a][1,4]-diazepin-2-yl]carbonyl]amino]propionicacid (L-738,167: The Journal of Pharmacology and ExperimentalTherapeutics, 281, 677-689, 1997), Intrifiban (Integrelin) (Circulation,94, 2083-2089, 1996) represented by the formula:

[0058] FK-633 (JP-A H5(1993)-148207) represented by the formula:

[0059] etc.

[0060] Further examples of the GPIIb/IIIa antagonists include2-piperazinone-1-acetic acid derivative (I) represented by the aboveformula (I) or a salt thereof, etc. Moreover, preferred examples of the2-piperazinone-1-acetic acid derivative include those described in WO96/33982 and, in particular, the 2-piperazinone-1-acetic acid derivative(I) represented by the above formula (I) wherein A¹ and A² areindependently (1) an amidino group or a guanidino group, each of whichmay be substituted with a C₂₋₈ alkoxycarbonyloxy group or a C₂₋₈alkoxycarbonyl group, (2) an amino group optionally having anoxadiazolyl group which may be substituted with a substituent selectedform an oxo group and an optionally halogenated C₁₋₄ alkyl group, or (3)an oxadiazolyl group which may be substituted with a substituentselected from an oxo group and an optionally halogenated C₁₋₄ alkylgroup [preferably, (1) an amidino group or a guanidino group, each ofwhich may be substituted with a methoxycarbonyloxy group or (2) an aminogroup optionally having a substituent selected from a5-oxo-1,2,4-oxadiazol-3-yl group and a5-trifluoromethyl-1,2,4-oxadiazol-3-yl group]; D is a group of theformula:

[0061] (wherein a is an integer of 0-2) [preferably, a phenylene group];R¹ is a hydrogen atom; R² is a hydrogen atom or a C₁₋₄ alkyl groupoptionally having a phenyl group which may be substituted with a C₁₋₄alkoxy group [preferably, a hydrogen atom or a p-methoxybenzyl group]; Pis a group of the formula: —Z—B— (wherein Z is a chemical bond, —NH— or—NH—CO— and B is a group of the formula:

[0062] or —(CH₂)_(c)— (wherein b is an integer of 0-1 and c is aninteger of 1-5)) [preferably, a group of the formula:

[0063] ]; Y is a group of the formula: —CO—R⁷ (wherein R⁷ is (1) ahydroxy group, (2) a C₁₋₈ alkoxy group or a C₂₋₁₂ alkenyloxy group, eachof which may be substituted with a substituent selected from a C₁₋₄alkoxy-carbonyl group or a 5-methyl-2-oxo-1,3-dioxolen-4-yl group, or(3) a group of the formula: —OCH(R^(7a))OCOR⁸ (wherein R^(7a) is ahydrogen atom or a C₁₋₆ alkyl group and R⁸ is a C₁₋₆ alkyl group or aC₅₋₇ cycloalkyloxy group)) [preferably, a carboxyl group]; and n is aninteger of 1-4 [preferably, an integer of 2-3]; is preferable.

[0064] More preferred examples of the 2-piperazinone-1-acetic acidderivative (I) include(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid,(S)-4-(4-amidinobenzoyl-amino)acetyl-3-[3-(4-amidinobenzoyl-amino)]propyl-2-oxopiperazine-1-aceticacid, or their hydrochloride, dihydrochloride, acetate, etc. Amongothers, hydrochloride of(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid is preferable and, in particular,(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoyl-amino)]propyl-2-oxopiperazine-1-aceticacid hydrochloride or(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid dihydrochloride is preferable. Said(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid dihydrochloride can be prepared by adding concentrated hydrochloricacid to a solution containing(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid hydrochloride and adjusting pH of the solution to about 1-2(preferably about 1.5), and the obtained(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid dihydrochloride can be crystallized with ethanol, etc.

[0065] Examples of the above-mentioned anti-tumor agents includebleomycin, methotrexate, actinomycin D, mitomycin C, vinblastinesulfate, vincristine sulfate, daunorbicin, adriamycin, neocarzinostatin,cytosine arabinoside, fluorouracil, tetrahydrofuryl-5-fluorouracil,krestin, picibanil, lentinan, levamisole, bestatin, glycyrrhizin,polynucleic acids such as poly IC, poly AU, poly ICLC, etc. [ImmuneResponse (Yuichi YAMAMURA and Seiji MORISAWA; 1977), page 143], etc.

[0066] Examples of the above-mentioned antibiotics include gentamycin,dibekacin, kanendomycin, lividomycin, tobramycin, amikacin, fradiomycin,sisomycin, tetracycline hydrochloride, oxytetracycline hydrochloride,rolitetracycline, doxycycline hydrochloride, ampicillin, piperacillin,ticarcillin, cefalotin, cefaloridine, cefotiam, cefsulodin, cefmenoxime,cefmetazole, cefazolin, cefotaxime, cefoperazone, ceftizoxime,moxolactam, thienamycin, sulfazecin, azusleonam, etc.

[0067] Examples of the above-mentioned antipyretics, analgesics andanti-inflammatory agents include salicylic acid, sulpyrine, flufenamicacid, diclofenac, indomethecin, morphine, pethidine hydrochloride,levorphanol tartarate, oxymorphone, etc.

[0068] Examples of the above-mentioned antitussive expectorants includeephedrine hydrochloride, methylephedrine hydrochloride, noscapinehydrochloride, codeine phosphate, dihydrocodeine phosphate, alloclamidehydrochloride, chlorphezianol hydrochloride, picoperidaminehydrochloride, cloperastine, protokylol hydrochloride, isoproterenolhydrochloride, salbutamol sulfate, terebutaline sulfate, etc.

[0069] Examples of the above-mentioned sedatives include chlorpromazine,prochlorperazine, trifluoperazine, atropine sulfate, methylscopolaminebromide, etc.

[0070] Examples of the above-mentioned muscle relaxants include pridinolmethanesulfonate, tubocurarine chloride, pancuronium bromide, etc.

[0071] Examples of the above-mentioned antiepileptic agents includephenytoin, ethosukimide, acetazolamide sodium, chlordiazepoxide, etc.

[0072] Examples of the above-mentioned antiulcer agent includemetoclopramide, histidine hydrochloride, etc.

[0073] Examples of the above-mentioned antidepressants includeimipramine, clomipramine, noxiptilin, phenelzine sulfate, etc.

[0074] Examples of the above-mentioned antiallergic agents includediphenhydramine hydrochloride, chlorpheniramine maleate, tripelennaminehydrochloride, clemizole hydrochloride, diphenylpyraline hydrochloride,methoxyphenamine hydrochloride, etc.

[0075] Examples of the above-mentioned cardiotonics includetransbioxocamphor, theophyllol, aminophylline, etilefrine hydrochloride,etc.

[0076] Examples of the above-mentioned antiarrhythmic agents includepropranolol, alprenolol, bufetolol, oxyprenolol, etc.

[0077] Examples of the above-mentioned vasodilators include oxyfedrinehydrochloride, diltiazem, tolazoline hydrochloride, hexobendine,bamethan sulfate, etc.

[0078] Examples of the above-mentioned hypotensive diuretics includehexamethonium bromide, pentolinium, mecamylamine hydrochloride,ecarazine hydrochloride, clonidine, etc.

[0079] Examples of the above-mentioned antidiabetic agents includeglymidine sodium, glipizide, phenformin hydrochloride, buforminhydrochloride, metformin, etc.

[0080] Examples of the above-mentioned anticoagulants include heparinsodium, sodium citrate, etc.

[0081] Examples of the above-mentioned hemostatics includethromboplastin, thrombin, menadione sodium bisulfite, acetomenaphthone,ε-aminocaproic acid, tranexamic acid, carbazochrome sodium sulfate,adrenochrome monoaminoguanidine methanesulfonate, etc.

[0082] Examples of the above-mentioned antituberculous agents includeisoniazid, ethambutol, para-aminosalicylic acid, etc.

[0083] Examples of the above-mentioned hormone preparations includeprednisolone, prednisolone sodium phosphate, dexamethasone sodiumsulfate, betamethasone sodium phosphate, hexoestrol phosphate,hexoestrol acetate, methimazole, etc.

[0084] Examples of the above-mentioned narcotic antagonists includelevallorphan tartarate, nalorphine hydrochloride, naloxonehydrochloride, etc.

[0085] Examples of the above-mentioned bone resorption inhibitorsinclude (sulfur-containing alkyl)aminomethylenebisphosphonic acid, etc.

[0086] Examples of the above-mentioned osteogenesis promoting agentsinclude vitamin K2 or parathyroid hormone, or a compound of the formula(II):

[0087] wherein ring A is an optionally substituted benzene ring, R is ahydrogen atom or an optionally substituted hydrocarbon group, B is anoptionally esterified or amidated carboxyl group, X is —CH(OH)— or —CO—,k is 0 or 1, and k′ is 0, 1 or 2, or a salt thereof, etc. (JP-AH3(1991)-232880, JP-A H4(1992)-364179).

[0088] Examples of the above-mentioned angiogenesis inhibitors includeangiostatic steroid [Science, 221, 719 (1983)], fumagillin[EP-A-325199], fumagillol derivatives [EP-A-357061, EP-A-359036,EP-A-386667, EP-A-415294], etc.

[0089] The physiologically active substance may be distinct entity or inthe form of any possible pharmaceutical salts thereof (e.g. a salt withan inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid,etc. or a salt with an organic salt such as carbonic acid, succinicacid, etc., when the physiologically active substance has a basic groupsuch as an amino group, etc.; a salt with an inorganic base such asalkaline metals (e.g. sodium, potassium, etc.), a salt with a basicorganic compound such as organic amines (e.g. triethylamine, etc.),basic amino acids (e.g. arginine, etc.), etc., when the physiologicallyactive substance has an acidic group such as carboxyl group, etc.). Whenthe physiologically active substance is distinct entity andwater-insoluble, it can be converted into a water-soluble salt thereof.

[0090] In the sustained-release microcapsule, the amount of thephysiologically active substance to be used varies with factors relatedto the particular kind of the physiologically active substance, desiredpharmacological activity, duration time, etc. The amount of thephysiologically active substance in the microcapsule ranges preferablyfrom about 0.01% to about 50% (W/W), more preferably from about 0.1% toabout 30% (W/W).

[0091] Examples of the biodegradable polymer to be used in the presentinvention include poly fatty acid esters (e.g. polylactic acid,polyglycolic acid, polycitric acid, polymalic acid, polylactic acidcaprolactone, etc.), poly-α-cyanoacrylic acid esters,poly-β-hydroxybutyric acids, polyalkylene oxalates (e.g.polytrimethyleneoxalate, polytetramethyleneoxalate, etc.),polyortho-esters, polyortho-carbonates, other polycarbonates (e.g.polyethylene-carbonate, polyethylene-propylene-carbonate, etc.),polyamino acids (e.g. poly-γ-benzyl-L-glutamic acid, poly-L-alanine,poly-γ-methyl-L-glutamic acid, etc.), hyaluronic acid esters, etc.

[0092] These polymers may optionally be used singly or as a copolymer oftwo or more of them or as a simple mixture of them or in the form oftheir salts.

[0093] The biodegradablity of these biodegradable polymer is defined asthe percentage (w/w %) of water-soluble low-molecular weight fragmentsdegraded from the polymer relative to the polymer when the polymer isused as injectable preparations, and in general, it should be not lessthan 10% in three months after subcutaneous or intramuscularadministration, preferably, not less than 80% in one year aftersubcutaneous or intramuscular administration.

[0094] Said biodegradable polymer is preferably aliphatic polyester.Examples of said biodegradable polymer include an aliphatic polyester (apoly fatty acid ester), more preferably, polymers or copolymers ofhydroxycarboxylic, or mixtures thereof.

[0095] The hydroxycarboxylic acids are not specifically limited, butpreferably hydroxycarboxylic acids of the formula:

[0096] wherein R is a hydrogen atom or an alkyl group.

[0097] In the above formula, examples of the alkyl group represented byR includes a straight or branched alkyl group having 1 to 8 carbon atoms(e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,pentyl, hexyl, heptyl, octyl, etc.). Among others, a straight orbranched alkyl group having 1 to 3 carbon atoms is more preferable.

[0098] Preferred examples of the above hydroxycarboxylic acid includeglycolic acid, lactic acid, hydroxybutyric acid (e.g. 2-hydroxybutyricacid), 2′-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid,2-hydroxycaproic acid, 2-hydroxyisocaproic acid, 2-hydroxycaprylic acid,etc. Among others, glycolic acid, lactic acid, 2-hydroxybutyric acid,2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid, etc. arepreferable. In particular, glycolic acid, lactic acid, 2-hydroxybutyricacid, etc. are more preferable. Where these hydroxycarboxylic acidsexist as D-isomers, L-isomers and D, L-isomers (racemic mixtures ofD-isomer and L-isomer), any one of them may be used. Preferably,D,L-isomers are used.

[0099] The copolymers may be any of random, block and graft copolymers.Preferred examples of the glycolic acid copolymer include the copolymerthat degrades in living body relatively rapidly and has a release periodof not more than one month when used alone. In particular, lacticacid/glycolic acid homopolymer or copolymer (hereinafter, referred to as“lactic acid/glycolic acid polymer” or abbreviated to “PLGA”, both ofwhich always include copolymers and homopolymers of the respectiveacids) or hydroxybutyric acid/glycolic acid homopolymer or copolymer(hereinafter, referred to as “hydroxybutyric acid/glycolic acidcopolymer”, which always include copolymers and homopolymers of therespective acids) is preferable.

[0100] The biodegradable polymer to be used in the present invention canbe synthesized by general synthetic methods (e.g. those described inJP-A S61(1986)-28521), without any problem.

[0101] In general, the weight-average molecular weight of thebiodegradable polymer to be used in the present invention rangespreferably from about 2,000 to about 800,000, more preferably from about5,000 to about 200,000. When lactic acid/glycolic acid copolymer is usedas the above-mentioned polymer, the molar ratio of lactic acid/glycolicacid ranges preferably from 100/0 to about 25/75, more preferably fromabout 100/0 to about 50/50. The weight-average molecular weight oflactic acid/glycolic acid copolymer ranges preferably about 5,000 toabout 30,000, more preferably from about 5,000 to about 20,000.

[0102] When hydroxybutyric acid/glycolic acid copolymer (e.g.2-hydroxybutyric acid/glycolic acid copolymer) is used as theabove-mentioned polymer, the molar ratio of hydroxybutyric acid/glycolicacid ranges preferably from about 100/0 to about 25/75, more preferablyfrom about 100/0 to about 50/50. In particular, the molar ratio of2-hydroxybutyric acid/glycolic acid ranges from preferably about 60/40to about 30/70. The weight-average molecular weight of hydroxybutyricacid/glycolic acid copolymer ranges preferably from about 5,000 to about25,000, more preferably from about 5,000 to about 20,000.

[0103] When butyric acid/glycolic acid copolymer is used as theabove-mentioned polymer, the molar ratio of butyric acid/glycolic acidranges preferably from about 100/0 to about 25/75.

[0104] When a mixture of polylactic acid (A) and glycolicacid/2-hydroxybutyric acid copolymer(B) is used as the above-mentionedpolymer, the mixing ratio of (A) /(B) ranges from about 10/90 to about90/10 (by weight), preferably from about 25/75 to about 75/25 (byweight). The weight-average molecular weight of polylactic acid rangespreferably from about 5,000 to about 30,000, more preferably from about6,000 to about 20,000.

[0105] In the present specification, the weight-average molecular weightmeans a molecular weight in terms of the molecular weight of polystyrenedetermined by gel permeation chromatography (GPC) using polystyrene asthe standard material. More specifically, the weight-average molecularweight is based on polystyrene, obtained by gel permeationchromatography (GPC) with 9 polymers of polystyrene as referencesubstances with weight-average molecular weights of 120,000, 52,000,22,000, 9,200, 5,050, 2,950, 1,055, 580 and 162, respectively. Thedetermination was carried out using GPC column KF804LX2 (manufactured byShowa Denko K. K., Japan) and RI monitor L-3300 (Hitachi, Japan) andusing chloroform as the mobile phase.

[0106] The polydispersity of said polymer is defined as the value ofweight average molecular weight/number average molecular weight, whichranges, in general, from 1 to 3.5, preferably from 1.5 to 2.5. Theamount of the biodegradable polymer to be used depends upon, forexample, the degree of the pharmacological activity of thephysiologically active substance, release rate of said substance,release period of said substance, etc. For example, the polymer is usedis used as the microcapsule base in an amount of about 0.2 to about10000 times (by weight), preferably about 1 to about 1000 times (byweight) relative to the weight of the physiologically active substance.The concentration of the biodegradable polymer in an oil phase rangespreferably from about 0.5 to about 90% (W/W), more preferably about 2 toabout 60% (W/W).

[0107] Examples of the “fat and oil” to be added to an organic solventof the above biodegradable polymer include any fat and oil which isdissolved in said organic solvent without causing phase separation andwhich is degradable and absorbable in living body, and preferablyexcluding a fatty acid, a salt thereof, a glycerin fatty acid ester anda propylene glycol fatty acid ester. Examples of the fat and oil includean oil soluble vitamin (vitamin A, vitamin D, vitamin E, vitamin K,etc.), medium chain fatty acid triglyceride (triglycerol of fatty acidhaving 8-12 carbon atoms such as miglyol, etc.), cholesterol,phospholipids, etc. Preferable examples of the fat and oil include anoil soluble vitamin (vitamin A, vitamin D, vitamin E, vitamin K, etc.),cholesterol, phospholipids, etc., more preferably an oil soluble vitaminsuch as α-tocopherol (vitamin E), α-tocopherol acetate (vitamin Eacetate), etc.

[0108] A final concentration of the fat and oil to be added to anorganic solvent of the above biodegradable polymer ranges from about 1%to about 50% (W/W), more preferably from about 3% to about 30% (W/W)relative to a whole content of the sustained-release microcapsules.

[0109] In the present invention, when the sustained-releasemicrocapsules are produced by a W/O technique or a W/O/W technique, abasic substance may be further added to an aqueous phase containing thephysiologically active substance. In particular, when thephysiologically active substance is an acidic drug or an acidic salt ofthe drug (hydrochloride, etc.) (for example, pH of the aqueous phase is6 or less), it is preferable to add the basic substance. Examples of thebasic substance include a basic amino acid such as L-histidine,L-arginine, L-lysine, etc., N-methylglucamine, etc.

[0110] The final concentration of the basic substance to be added to thesolution of the physiologically active substance ranges from about 0.1%to about 20% (W/W) relative to a whole content of the sustained-releasemicrocapsule, more preferably about 1% to about 8% (W/W). In the presentinvention, it is preferable to allow an osmotic pressure adjustor to becontained in the outer aqueous phase when removal of the organic solventis carried out by in-water drying.

[0111] Any osmotic pressure adjustor can be employed so long as itproduces osmotic pressure in an aqueous solution thereof.

[0112] Examples of the osmotic pressure adjustor include water-solublepolyhydric alcohols, water-soluble monovalent alcohols, water-solubleinorganic materials (e.g. inorganic salts), water-solublemonosaccharides, disaccharides, oligosaccharides polysaccharides or itsderivative, water-soluble organic acids or a salt thereof, water-solubleamino acid, water-soluble peptide, protein or its derivative, etc. Amongothers, water-soluble polyhydric alcohols, water-soluble inorganicmaterials, water-soluble monosaccharides, disaccharides,oligosaccharides polysaccharides or its derivative, water-solubleorganic acid or a salt thereof, etc. are preferable. In particular,salts, water-soluble polyhydric alcohols and water-soluble inorganicmaterials are preferable.

[0113] Examples of the above water-soluble inorganic salts includehalogenated alkali metals such as potassium chloride, sodium chloride,potassium bromide, sodium bromide, potassium iodide, sodium iodide,etc., halogenated alkaline earth metals such as calcium chloride,magnesium chloride, etc., alkali metal sulfates such as sodium sulfate,potassium sulfate, etc., alkaline earth metal sulfates such as magnesiumsulfate, calcium sulfate, etc., alkali metal phosphates such aspotassium dihydrogenphosphate, dipotassium hydrogenphosphate, potassiumphosphate, sodium dihydrogenphosphate, disodium hydrogenphosphate,sodium phosphate, etc., etc. Among others, sodium chloride ispreferable.

[0114] Examples of the above water-soluble polyhydric alcohol includedihyrdic alcohols such as glycerin, etc., pentahydric alcohols such asarabitol, xylitol, adonitol, etc., hexahydric alcohols such as mannitol,sorbitol, etc., etc. Among others, hexahydric alcohols are preferable.Examples of the above water-soluble monovalent alcohol include methanol,ethanol, isopropyl alcohol, etc. Among others, ethanol is preferable.Examples of the above water-soluble monosaccharides include pentosessuch as arabinose, xylose, ribose, 2-deoxyribose, etc., hexoses such asglucose, fructose, galactose, mannose, sorbose, rhamnose, fucose, etc.Among others, hexoses are preferable.

[0115] Examples of the above water-soluble disaccharides includemaltose, cellobiose, α-trehalose, lactose, sucrose, etc. Among others,lactose and sucrose are preferable.

[0116] Examples of the above water-soluble oligosaccharides includetrisaccharides such as maltotriose, raffinose, etc., tetrasaccharidessuch as stachyose, etc., etc. Among others, trisaccharides arepreferable.

[0117] Examples of the above water-soluble polysaccharides includeglucans such as cellulose, starch, glycogen, etc., galacturonans such aspectic acid, etc., mannuronans such as alginic acid, etc., fructans suchas inulin, levan, etc., N-acetylglycosamine polymers, such as chitin,etc., xylans such as xylan of rice straw, etc., diheteroglucans such asmannan, glucomannan, galactomannan, hyaluronic acid, chondroitinsulfate, heparin, etc., etc. Among others, glucans, diheteroglucans,etc. are preferable.

[0118] Examples of the derivatives of the above water-solublemonosaccharides, disaccharides, oligosaccharides and polysaccharidesinclude glucosamine, galactosamine, glucuronic acid, galacturonic acid,etc. Examples of the above water-soluble organic acid or a salt thereofinclude citric acid, tartaric acid, malic acid, their alkali metal salt(e.g. sodium salt, potassium salt, etc.), etc. Examples of the abovewater-soluble amino acid include neutral amino acid such as glycine,alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,tryptophan, serine, threonine, proline, hydroxyproline, cyctein,methionine, etc., acidic amino acid such as aspartic acid, glutamicacid, etc., basic amino acid such as lysine, arginine, histidine, etc.,etc. Salts of these water-soluble amino acid with acids (e.g.hydrochloric acid, sulfuric acid, phosphoric acid, etc.) or alkalis(e.g. alkali metal such as sodium, potassium, etc., etc.) are also usedoptionally. Examples of the water-soluble peptide, protein or theirderivative include casein, globulin, prolamin, albumin, gelatin,protamine, histone, etc.

[0119] These osmotic pressure adjustor may be used alone or as a mixtureof two or more of them. When the osmotic pressure adjustor is anon-ionic material, the concentration of the osmotic pressure adjustorin the outer aqueous phase ranges from about 0.001 to about 60% (W/W),preferably from about 0.01 to about 40% (W/W), more preferably fromabout 0.05 to about 30% (W/W). When the osmotic pressure adjustor is anionic material, it is used in a concentration calculated by dividing theabove-mentioned concentration by the total ionic valency. Theconcentration of the osmotic pressure adjustor to be added is notnecessarily below their solubility, and a part of it may be left in thestate of dispersion in the solvent.

[0120] Each step of the production method of the present invention iscarried out, for example, as follows:

[0121] In the process for producing the microcapsule where a solution ofthe water-soluble physiologically active substance is used as an aqueousphase, the water-soluble physiologically active substance (hereinafter,briefly referred to as the drug) is dissolved in water, and ifnecessary, basic substances such as above-mentioned basic amino acid,etc. and additionally pharmaceutical carriers such as e.g. gelatin,agar, polyvinylalcohol, etc. are added to the solution to prepare anaqueous phase (an inner aqueous phase).

[0122] The concentration of the drug in the inner aqueous phase rangespreferably from about 0.1 to about 150% (W/V), more preferably about 20to about 130% (W/V), and in particular, about 60 to about 120% (W/V).

[0123] As pH regulators to maintain the stability and solubility of thedrug, for example, carbonic acid, acetic acid, oxalic acid, citric acid,phosphoric acid, hydrochloric acid, sodium hydroxide, arginine, lysineand their salts, etc. can be added to said aqueous phase. Further, asstabilizers of the drug, there can be added, for example, albumin,gelatin, citric acid, sodium ethylenediaminetetraacetate, dextrin,sodium hydrogensulfite, polyols such as polyethyleneglycol, etc., etc.,and as preservatives there can be added, for example, conventionalpara-oxybenzoic acid esters (e.g. methylparaben, propylparaben, etc.),benzylalcohol, chlorobutanol, thimerosal, etc.

[0124] The thus obtained aqueous phase is added to a uniform organicsolvent solution (an oil phase) containing the biodegradable polymer(hereinafter, briefly referred to as the polymer) and a fat and oil,followed by emulsification to prepare a W/O type emulsion. Theemulsification can be carried out by conventional dispersion techniquessuch as intermittent shaking, mixing by means of a mixer (e.g. propelleragitator, turbine agitator, etc.), colloid mill operation, mechanicalhomogenization, ultrasonication, etc.

[0125] The above polymer solution (oil phase) can be prepared bydissolving the polymer in an organic solvent which does notsubstantially mix with water. The water-solubility of said organicsolvent is preferably not more than 3% (W/W) at standard temperature(20° C.), and the boiling point of said organic solvent is preferablynot more than 120° C. Examples of the organic solvent includehalogenated hydrocarbons (e.g. dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride, etc.), alkyl ethershaving 3 or more carbon atoms (e.g. isopropylether, etc.), fatty acidalkyl (having 4 or more carbon atoms) esters (e.g. butyl acetate, etc.),aromatic hydrocarbons (e.g. benzene, toluene, xylene, etc.), etc. Thesesolvents can be used alone or in combination thereof. As the organicsolvent, halogenated hydrocarbons (e.g. dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride, etc.) are morepreferable, and in particular, dichloromethane is preferable.

[0126] Removal of the organic solvent from the thus produced W/O typeemulsion can be carried out by a conventional method. Examples of theremoval method of the organic solvent include spray drying, in-waterdrying, etc., preferably in-water drying.

[0127] In the spray drying technique, the produced W/O type emulsion isejected in a mist form through a nozzle, etc. into the drying chamber ofa spray drier to evaporate the solvent from the finely-divided liquiddroplets in a brief time. Examples of the nozzle include a two-fluidnozzle, pressure nozzle, rotary disk nozzle, etc.

[0128] In the in-water drying, the produced W/O type emulsion is addedto an aqueous phase (an outer aqueous phase) to form a W/O/W typeemulsion, followed by removing the organic solvent in the oil phase. Thevolume of the outer aqueous phase is generally selected from the rangeof about 1 to about 10,000 times the volume of the oil phase, morepreferably about 2 to about 5,000 times, and in particular, about 5 toabout 2,000 times.

[0129] Any emulsifier may be added to the above outer aqueous phase, aslong as it can contribute to the formation of a stable W/O/W typeemulsion. Examples of the emulsifiers include anionic surfactants(sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), non-ionicsurfactants (polyoxy-ethylene-sorbitan fatty acid esters [Tween 80,Tween 60; Atlas Powder], polyoxyethylene-castor oil derivatives [HCO-60,HCO-50; Nikko Chemicals], etc.), polyvinylpyrrolidone, polyvinylalcohol,carboxymethylcellulose, lecithin, gelatin, hyaluronic acid, etc.,preferably polyvinylalcohol. These emulsifiers can be used independentlyor in combination. The concentration may be selected from about 0.001 toabout 20% (W/W), more preferably about 0.01 to about 10% (W/W), and inparticular, from about 0.05 to about 5% (W/W).

[0130] The above-mentioned osmotic pressure adjustor may be added to anouter aqueous phase. In the production method of the present invention,it is preferable to adjust viscosity of the W/O type emulsion in therange of from about 150 centipoise (cp) to about 10,000 cp when theW/O/W type emulsion is formed.

[0131] Examples of the methods to adjust viscosity include (1) a methodfor adjusting the concentration of the biodegradable polymer in oilphase, (2) a method for adjusting ratio between an amount of the aqueousphase and that of the oil phase, (3) a method for adjusting thetemperature of the W/O type emulsion, (4) a method for adjusting thetemperature of the outer aqueous, (5) a method for adjusting thetemperature of the W/O type emulsion with line heater, cooler, etc. whenthe W/O type emulsion is injected. Each of these methods may be employedalone or in combination with each other.

[0132] Any of the above methods may be employed, as long as viscosity ofthe W/O type emulsion is in the range of from about 150 cp to about10,000 cp when the W/O type emulsion is changed into the W/O/W typeemulsion.

[0133] In the above method (1), the concentration of the bio-degradablepolymer in oil phase to be adjusted varies depending on the kind of thebiodegradable polymer, the kind of the organic solvent, etc., can not bedefined as restricted one, and ranges preferably from about 10 to about80% (W/W).

[0134] In the above method (2), ratio between an amount of the aqueousphase and that of the oil phase to be adjusted varies depending on thekind and amount of the drug, the character of the oil phase, etc., cannot be defined as restricted one, and ranges preferably W/O=about 1% toabout 50% (V/V).

[0135] In the above method (3), the temperature of the W/O type emulsionto be adjusted ranges, for example, from about −20° C. to the boilingpoint of the organic solvent, preferably form about 0° C. to about 30°C., more preferably from about 10° C. to about 20° C.

[0136] Adjusting viscosity of the W/O type emulsion in the above methods(1) and (2) can be carried out when the W/O type emulsion is produced.

[0137] In the above method (4), before adding the W/O type emulsion tothe outer aqueous phase, the temperature of the outer aqueous phase canbe adjusted to obtain a similar result in the above method (3). Thetemperature of the outer aqueous phase ranges, for example, from about5° C. to about 30° C., preferably from about 10° C. to about 25° C.,more preferably from about 12° C. to about 20° C.

[0138] The removal of the organic solvent can be carried out byconventional methods. For example, it is carried out by evaporating theorganic solvent by stirring with a propeller-type stirrer, magneticstirrer, etc. under atmospheric pressure or gradually reducing pressureor while controlling degree of vacuum by using a rotary evaporator,etc., etc.

[0139] When a water insoluble metal salt (or a metal salt which ishardly soluble in water) of the physiologically active peptide is usedas a physiologically active substance, a dispersion which is obtained bydispersing the physiologically active peptide metal salt in the organicsolvent containing the biodegradable polymer and the fat and oil ismixed well to give an organic solvent dispersion (hereinafter, referredto as a S/O type emulsion for the sake of convenience) whose stabilityof dispersion is high and where the physiologically active peptide metalsalt is substantially uniformly dispersed or suspended in the organicsolvent.

[0140] Examples of the above organic solvents include a similar organicsolvent to the organic solvent which is used for the preparation of theoil phase containing the biodegradable polymer and the fat and oil inthe preparation of the above W/O type emulsion.

[0141] For the preparation of the above S/O type emulsion, conventionaldispersion techniques can be employed. Examples of the dispersiontechniques include intermittent shaking, mixing by means of a mixer(e.g. propeller agitator, turbine agitator, etc.), colloid milloperation, mechanical homogenization, ultrasonication, etc.

[0142] In this case, it is useful to use a water-soluble solventtogether with a water-insoluble solvent, if desired. As saidwater-soluble solvent, any solvent can be employed as long as it iswater-soluble and can mix with the above water-insoluble solvent.Examples of said water-soluble solvent include alcohols (e.g. methanol,ethanol, propyl alcohol, isopropyl alcohol, etc.), acetone,acetonitrile, etc.

[0143] In the preparation of the s/o type emulsion, the physiologicallyactive substance is preferably finely pulverized to microparticles anddispersed in oil phase. The pulverized particle size ranges usually fromabout 1 nm to about 30 μm, preferably about 1 nm to about 5 μm, morepreferably about 1 nm to about 1 μm.

[0144] Subsequently, the S/O type emulsion is subjected to removal ofthe organic solvent according to a similar method applied for the aboveW/O type emulsion, and in-water drying is preferably employed.Preferably, an osmotic pressure adjustor is allowed to be contained inthe outer aqueous phase in the above-mentioned concentration. Morespecifically, said oil phase is added to the second aqueous phasecontaining the osmotic pressure adjustor to form an S/O/W type emulsion,followed by removing the organic solvent in the oil phase to preparemicrocapsules.

[0145] To the outer aqueous phase in the S/O/W type in-water dryingmethod, emulsifiers may be added. Examples of the emulsifiers include asimilar emulsifier to those described for the preparation of the aboveW/O/W type emulsion.

[0146] The removal of the organic solvent in the oil phase can becarried out by conventional methods. For example, it is carried out byevaporating the organic solvent by stirring with a propeller-typestirrer, magnetic stirrer, etc. under atmospheric pressure or graduallyreducing pressure or while controlling degree of vacuum by using arotary evaporator, etc., etc. In this case, at the time whensolidification of the polymer proceeds in some degree and the losscaused by the release of the physiologically active substance from theinternal phase is decreased, the S/O/W type emulsion may be warmedgradually to remove the organic solvent more completely, which resultsin saving of the required time. Alternatively, when thickening andsolidification are carried out by methods other than those based ontemperature, the removal can be carried out by merely allowing the S/O/Wtype emulsion to stand with stirring, by warming it, by sprayingnitrogen gas, etc., etc.

[0147] This removal step of the organic solvent is of importance andgreatly influences the surface structure of microcapsule controlling therelease of the physiologically active substance. For example, rapidremoval produces a number of pores on the surface or makes pores largerin their size, which results in increased release rate of thephysiologically active substance.

[0148] The thus obtained microcapsules are collected by centrifugationor filtration. Then, free physiologically active substance, carrierstherefor, etc. attached onto the surface of the microcapsules are washedwith distilled water repeatedly several times, and water and the solventin the microcapsules are removed more completely in reduced pressure, ifnecessary, with warming.

[0149] The thus obtained microcapsules are usually dispersed indistilled water, etc., subjected to freeze-drying and stored. In freezedrying, aggregation inhibitors may be added. Examples of saidaggregation inhibitors include water-soluble polysaccharides (e.g.mannitol, etc.), starch (e.g. corn starch, etc.), inorganic salts, aminoacids, proteins, etc. Among others, mannitol is preferable. Mixing ratio(by weight) between the microcapsules and the aggregation inhibitorsranges about 50:1 to about 1:1, preferably about 20:1 to about 1:1, morepreferably about 10:1 to about 5:1. In order to prevent the particlesfrom aggregating with each other while washing, aggregation inhibitorsmay be added to distilled water as washing solution. Examples of saidaggregation inhibitors include water-soluble polysaccharides such asmannitol, lactose, glucose, etc., amino acids such as glycine, etc.,proteins such as fibrin, collagen, etc., inorganic salts such as sodiumchloride, sodium hydrogen phosphate, etc., etc., preferably mannitol.

[0150] After freeze drying, removal of water and an organic solvent maybe carried out by warming the microcapsules under reduced pressure, ifdesired. When warming temperature is less than glass transitiontemperature of the biodegradable polymer used as a base, improvement ofinitial release of an excess amount of the physiologically activepeptide is not expected. When warming temperature is too high, thereincreases a danger of melting the microcapsules and attaching to eachother, deformation of the microcapsules, decomposition of thephysiologically active substance, decrease of activity of thephysiologically active substance, etc. In general, warming temperatureis selected appropriately, considering properties of the biodegradablepolymer used as a base (e.g. molecular weight, stability, etc.), kind ofthe physiologically active peptide, average particle size of themicrocapsules, warming time, degree of dryness of the microcapsules,warming method, etc. Preferably, the microcapsules are warmed and driedat an appropriate temperature which is not less than glass transitiontemperature of the biodegradable polymer used as a base and whereparticles of the microcapsules do not attached to each other. Inparticular, the microcapsules are warmed and dried at temperaturesranging from glass transition temperature to about 30° C. higher thanglass transition temperature of the biodegradable polymer used as abase. In this specification, glass transition temperature is defined bymedian glass transition temperature which is determined usingdifferential scanning calorimeter at temperature increments of 10 or 20°C. per minute.

[0151] Warming and drying time varies depending on warming temperature,an amount of the microcapsules to be treated, etc. In general, about 24to about 120 hours after temperature of microcapsules themselves reachto the desired temperature are preferable. In particular, about 48 toabout 120 hours are preferable.

[0152] A method for warming the microcapsules is not limited to aspecific method, and any method can be employed as long as themicrocapsule are uniformly warmed.

[0153] The microcapsules thus obtained are screened, and if necessary,after light pulverization, too large microcapsules are removed bysieving. Particle size of the microcapsules varies with the desireddegree of sustained release. When the microcapsules are used assuspension, particle size of the microcapsules are selected from therange satisfying the dispersibility and needle-pass requirements. Forexample, the average diameter ranges preferably from about 0.5 to 400μm, more preferably about 2 to 200 μm.

[0154] In order to prepare a sterile preparation of the microcapsules, amethod for sterilizing all production steps, a method for sterilizingwith γ-rays, a method for adding a preservative, etc. are employed, andthere is no limitation to a specific method.

[0155] The microcapsules prepared according to the process of thepresent invention can be readily administered orally or non-orally, forexample, as injections or implants intramuscularly, subcutaneously, intoblood vessels, organs, cava articulare, foci, etc. Further, they can beadministered in the form of various preparations. They can also be usedas raw materials in the production of various preparations.

[0156] Examples of the above preparation include injections, oralpreparations (e.g. powders, granules, capsules, tablets), nasalpreparations, suppositories (e.g. rectal suppositories, vaginalsuppositories), etc.

[0157] These preparations can be produced according to conventionalmethods. For example, when the microcapsules according to the presentinvention are to be processed into injections, the microcapsules of thepresent invention are dispersed in a aqueous vehicle together with adispersing agent (e.g. Tween 80, HCO 60 (Nikko Chemicals),carboxymethylcellulose, sodium arginate, etc.), a preservative (e.g.methylparaben, propylparaben, benzylalcohol, chlorobutanol, etc.), atonicity agent (e.g. sodium chloride, glycerin, sorbitol, glucose,etc.), etc., or an oily vehicle such as vegetable oils (e.g. olive oil,sesame oil, peanut oil, cotton-seed oil, corn oil, etc., propyleneglycol, etc., to prepare sustained-release injections of themicrocapsules. Further, excipients (e.g. mannitol, sorbitol, lactose,glucose, etc.) may be added, in addition to the above components, to theabove sustained-release injections of the microcapsules in the form ofsuspensions. After redispersion, the injections are solidified by freezedrying or spray drying, and distilled water for injection or anappropriate dispersing agent may be added just before use. In thismanner, more stable sustained-release injections can be obtained.

[0158] When the microcapsules according to the present invention are tobe processed into, for example, tablets, they can be prepared accordingto conventional preparation methods. For example, there can be addedexcipients (e.g. lactose, crystalline cellulose, sucrose, starch such ascorn starch, etc., etc.), disintegrating agents (e.g. starch such ascorn starch, etc., croscarmellose sodium, carboxymethylstarch sodium,calcium carmonate, etc.), binders (e.g. crystalline cellulose, gumarabic, dextrin, carboxymethylcellulose, polyvinylpyrrolidone,hydroxypropylcellulose, etc.), lubricants (e.g. talc, magnesiumstearate, polyethylene glycol 6000, etc.), etc. to the microcapsules,and the mixture is compressed for molding.

[0159] When the microcapsules according to the present invention are tobe processed into, for example, nasal preparations, they are molded intosolid, semisolid or liquid. In any case, conventional preparationmethods can be used. For example, to prepare the above solid nasalpreparations, said microcapsules either as they are or together withexcipients (e.g. glucose, mannitol, starch, microcrystalline cellulose,etc.), thickeners (e.g. natural gums, cellulose derivatives,polyacrylates, etc.), etc. are processed into powdery compositions. Toprepare the above liquid nasal preparations, said microcapsules areprocessed into an oily or aqueous suspension in substantially the samemanner as injections. The semisolid nasal preparations may be oily oraqueous gels or ointments. In any case, there may be added a pH adjustor(e.g. carbonic acid, phosphoric acid, citric acid, hydrochloric acid,sodium hydroxide, etc.), a preservative (e.g. paraoxybenzoic acidesters, chlorobutanol, benzalkoniumchloride, etc.), etc.

[0160] When the microcapsules according to the present invention are tobe processed into suppositories, they can be prepared per se knownmethods in the form of oily or aqueous solid, semisolid or liquid, withusing an oily or aqueous base. Examples of the oily base to be used forthe above composition include any one which does not dissolve themicrocapsules, for example, higher fatty acid glycerides [e.g. cacaobutter, Witepsols (Dynamit-Nobel), etc.], medium chain fatty acidtriglycerides [e.g. miglyols (Dynamit-Nobel), etc.], vegetable oils(e.g. sesame oil, soybean oil, cottonseed oil, etc.), etc. Examples ofthe aqueous base include polyethyleneglycols, propyleneglycols, etc.Examples of the aqueous gels include natural gums, cellulosederivatives, vinyl polymers, polyacrylates, etc.

[0161] The microcapsules obtained according to the production method ofthe present invention suppress initial burst and release a constantamount of the drug for a long time. Therefore, the microcapsulesobtained according to the production method of the present inventionexhibits a constant efficacy with low toxicity, thus being expected as asafe and highly effective sustained-release preparation. In addition,when the microcapsules are applied for the treatment of chronic disease,physical burden can be lightened and compliance can be improved in apatient who has to receive frequent administrations.

[0162] For example, in case of pituitary dwarfism, administration ofgrowth hormone is inevitable and so far growth hormone is subcutaneouslyor intramuscularly administered to infants or juvenile patients everyday or every second day for several months to 10 years or more. Sincethe sustained-release microcapsule obtained by the production method ofthe present invention provide these patients with sufficient treatmenteffect with one administration within several weeks to months, it cancontribute to improvement of compliance in these patients.

[0163] For example, when the sustained-release microcapsule of thepresent invention containing a water-soluble physiologically activesubstance which is an antithrombus agent is used for the treatment ofthrombosis, the sustained-release microcapsule of the present inventioncan stably maintain blood level of the drug within an effective dosewhich does not cause any side effect for a long time, while there isfear that a conventional method for administering an antithrombus agentcauses bleeding as side effect of antithrombus activity. Therefore, thesustained-release microcapsule of the present invention can bepositively used not only for the treatment of said chronic disease for along time but also for the prevention of the same. Thus, themicrocapsule of the present invention shows less side effect and lowtoxicity, and therefore, can be safely administered to warm bloodedmammal such as mouse, rat, dog, horse, bovine, human, etc.

[0164] While the dosage of the sustained-release microcapsule varieswith the types and content of the physiologically active substance asthe principal ingredient, dosage forms, duration of the release of thedrug, subject animals, purposes of administration, etc., it issufficient if only the principal ingredient is contained in an effectiveamount.

[0165] For example, when the sustained-release microcapsule of thephysiologically active peptide or a salt thereof is 1-week typesustained-release preparation, the unit dosage for an adult is selectedfrom the range preferably from about 0.0001 to 10 mg/kg, more preferablyabout 0.0005 to 1 mg/kg.

[0166] Number of administrations can be appropriately selected from oncea week, once per two weeks, once a month, etc., depending on the typesand content of the physiologically active substance, dosage forms,duration of the release of the drug, subject diseases, subject animals,etc.

[0167] When the physiologically active polypeptide as active ingredientof the sustained-release preparation is human growth hormone and 2-weektype preparation of the sustained-release preparation is administered toa patient of pituitary dwarfism, the unit dosage of the activeingredient is usually selected from about 0.01 to about 5 mg/kg,preferably about 0.03 to about 1 mg/kg, and sustained-releasepreparation is administered once per two weeks. When the physiologicallyactive substance is insulin, the unit dosage of the active ingredient toa patient of diabetes is usually selected from about 0.001 to about 1mg/kg, preferably about 0.01 to about 0.2 mg/kg, and sustained-releasepreparation is administered once a week.

[0168] When the sustained-release microcapsule containing2-piperazinone-1-acetic acid derivative or a salt thereof represented bythe above formula (I) which is anticoagulant is orally administered to apatient of unstable angina, or a patient of ischemic complication orreobstruction of coronary or restenosis of coronary after PTCA orcoronary thrombolytic therapy, the unit dosage for an adult (bodyweight: 50 kg) is selected from about 1 mg to 10 g, preferably about 1mg to 2 g as an amount of the microcapsule (usually, about 1 to 500 mg,preferably about 10 to 200 mg as an amount of Compound (I) as theprincipal ingredient).

[0169] When the sustained-release microcapsule containing2-piperazinone-1-acetic acid derivative or a salt thereof represented bythe above formula (I) is non-orally administered to a patient ofunstable angina, or a patient of ischemic complication or reobstructionof coronary or restenosis of coronary after PTCA or coronarythrombolytic therapy. The unit dosage for an adult (body weight: 50 kg)is selected from about 0.1 to 5 ml, preferably about 0.5 to 3 ml as avolume of a suspension to be administered as injection (usually, about0.05 to 50 mg, preferably 1 to 20 mg as an amount of Compound (I) as theprincipal ingredient).

[0170] The sustained-release microcapsule obtained by the productionmethod of the present invention is stored at standard temperatures or ata cold place, preferably at a cold place. In this specification,standard temperature or a cold place is the same as that defined in ThePharmacopoeia of Japan. That is, standard temperature is 15-25° C., anda cold place is the place where the temperature is 15° C. or less.

EXAMPLES

[0171] The present invention is hereinafter described in more detail bymeans of, but not limited to, the following Working Examples and TestExamples.

Working Example 1

[0172] In distilled water 1 ml were dissolved(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid (hereinafter, briefly referred to as Compound A) hydrochloride 500mg and L-arginine 150 mg to give an inner aqueous phase. In methylenechloride 7.5 ml were dissolved lactic acid/glycolic acid copolymer(lactic acid/glycolic acid=50/50 (mole %), weight-average molecularweight 8,000) 3850 mg and vitamin E 500 mg to give an oil phase. The oilphase was added to the inner aqueous phase, and the mixture wasemulsified with small homogenizer (Polytron) to give a W/O typeemulsion. The W/O emulsion was emulsified in 0.1% PVA solution 800 ml(an outer aqueous phase) containing 2.7% NaCl which was cooled to 15° C.with using homomixer to give a W/O/W type emulsion. Then the W/O/W typeemulsion was slowly stirred with a conventional propeller agitator for 3hours. After hardening of the microcapsules with evaporation ofmethylene chloride, the microcapsules were collected by centrifugation.The collected microcapsule were washed with purified water and subjectedto freeze drying. The microcapsule 20 mg was dissolved inacetonitrile/distilled water (2:1 mixture) 3 ml. To the mixture wasadded 0.5 N KOH-ethanol solution 3 ml, and the mixture was subjected tohydrolysis at 25° C. for 20 hours, followed by evaporation undernitrogen gas stream. The residue was neutralized with 0.5 N HCl, and thesolution was diluted with 0.01 N HCl to give a solution of a final aminoacid concentration at about 200 nmol/ml and subjected to amino acidanalyzer (Hitachi L-8500A) to determine the content of arginine. In themicrocapsules, the content of arginine was 1.6%, and the content ofvitamin E in the microcapsules determined according to a methoddescribed in Working Example 5 shown below was 10%.

Working Example 2

[0173] In distilled water 1 ml were dissolved Compound A hydrochloride500 mg and L-arginine 150 mg to give an inner aqueous phase. Inmethylene chloride 8 ml were dissolved lactic acid/glycolic acidcopolymer (lactic acid/glycolic acid=50/50 (mole %), weight-averagemolecular weight: 8,000) 4100 mg and vitamin E 250 mg to give an oilphase. The oil phase was added to the inner aqueous phase, and themixture was emulsified with small homogenizer (Polytron) to give a W/Otype emulsion. The W/O emulsion was emulsified in 0.1% PVA solution 800ml (an outer aqueous phase) containing 2.7% NaCl which was cooled to 15°C. with using homomixer to give a W/O/W type emulsion. Then, the W/O/Wtype emulsion was slowly stirred with a conventional propeller agitatorfor 3 hours. After hardening of the microcapsules with evaporation ofmethylene chloride, the microcapsules were collected by centrifugation.The collected microcapsules were washed with purified water, to whichwas added mannitol 440 mg, and subjected to freeze drying.

Working Example 3

[0174] In distilled water 2 ml were dissolved Compound A hydrochloride750 mg and L-arginine 150 mg to give an inner aqueous phase. Inmethylene chloride 10 ml were dissolved lactic acid/glycolic acidcopolymer (lactic acid/glycolic acid=50/50 (mole %), weight-averagemolecular weight: 9,000) 3600 mg and vitamin E 500 mg to give an oilphase. According to a similar method described in Working Example 2 theW/O type emulsion was prepared, and thereafter the W/O/W type emulsionwas prepared, and finally freeze dried microcapsules was prepared. Inthe prepared microcapsules, content of arginine and vitamin E wasrespectively 1.5% (w/w) and 10% (w/w).

Working Example 4

[0175] In distilled water 1 ml were dissolved Compound A hydrochloride500 mg and L-arginine 150 mg to give an inner aqueous phase. Inmethylene chloride 8 ml were dissolved lactic acid/glycolic acidcopolymer (lactic acid/glycolic acid=50/50 (mole %), weight-averagemolecular weight: 9,000) 4100 mg and vitamin E 250 mg to give an oilphase. According to a similar method described in Working Example 2 theW/O type emulsion was prepared, and thereafter the W/O/W type emulsionwas prepared, and finally freeze dried microcapsules was prepared. Inthe prepared microcapsules, content of arginine and vitamin E wasrespectively 1.8% (w/w) and 5.2% (w/w).

Comparative Example 1

[0176] In distilled water 0.5 ml was dissolved Compound A 200 mg to givean inner aqueous phase. In methylene chloride 2 ml was dissolved lacticacid/glycolic acid copolymer (lactic acid/glycolic acid=50/50 (mole %),weight-average molecular weight 9,000) 1800 mg to give an oil phase.According to a similar method described in Working Example 3 the W/Otype emulsion was prepared, and thereafter the W/O/W type emulsion wasprepared, and finally freeze dried microcapsules was prepared.

[0177] In Table 1, properties of the microcapsules obtained in WorkingExample 4 and Comparative Example 1 are shown. TABLE 1 Initial Releaseof 1 day Method Entrapment in vitro Release Test Working Example 4 96%10% Comparative Example 1 88% 68%

[0178] As shown in Table 1, entrapment ratio of the drug into themicrocapsules was increased and initial release of 1 day with in vitroRelease Test was suppressed according to the production method of themicrocapsules characterized by adding 3% arginine to an inner aqueousphase and 5% vitamin E to an oil phase with using Compound Ahydrochloride.

Test Example 1

[0179] The microcapsules (20 mg/kg as the drug content) obtained in theabove Working Example 1 and Comparative Example 1 were subcutaneouslyadministered to SD rats (male, 6 weeks) and the plasma level of the drugafter administration was determined by ELISA, respectively. The thusobtained results were shown in FIG. 1. In FIG. 1, the curve A shows theplasma level of the drug administered by the microcapsules of WorkingExample 1 and the curve B shows the plasma level of the drugadministered by the microcapsules of Comparative Example 1. The plasmalevel of the drug 1 hour after administration of the microcapsules ofWorking Example 1 and Comparative Example 1 were respectively 689 ng/mland 2926 ng/ml. Therefore, it became possible that increase of theplasma level of the drug was suppressed at an early stage afteradministration, the time-course changes of the plasma level of the drugwas not remarkable and the effective plasma level of the drug was stablymaintained for about 2 weeks in the microcapsules prepared by adding 3%arginine to an inner aqueous phase and 10% vitamin E to an oil phasewith using Compound A hydrochloride.

Reference Example 1

[0180] To a solution (710 mg/355 ml, 5 mM NH₄HCO₃, pH 7.8) containingrecombinant human growth hormone having methionine at its amino terminal(prepared according to the method described in Reference Example 14 ofJP-A S62(1987)-171699) [hereinafter, briefly referred to as Met-hGH] wasgradually added dropwise Zn(OAc)₂ 2H₂O (71 mg/5 ml H₂O) solution 3.5 ml(Met-hGH:Zn=1:7 mole) while stirring. The resulting insoluble Met-hGH/Zncomplex was centrifuged at 2,500 rpm for 20 minutes, and the supernatantwas removed. The insoluble Met-hGH/Zn complex was redispersed indistilled water 100 ml and subjected to freeze drying.

Working Example 5

[0181] In a mixture of methylene chloride 2.0 ml and acetonitrile 0.4 mlwere dissolved lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50 (mole %), weight-average molecular weight: 15,000; Wako PureChemical) 1275 mg and vitamin E 150 mg to give an oil phase. In the oilphase was dispersed Met-hGH/Zn complex 75 mg obtained in ReferenceExample 1. The dispersion was subjected to ultrasonication for 5 minutesand then to a small homogenizer (Polytron) at 15,000 rpm for 1 minute topulverize it to microparticles. The S/O dispersion was added topolyvinylpyrrolidone (PVA) solution 800 ml containing 10% mannitolcooled at 15° C. and emulsified with homomixer to give a S/O/W typeemulsion. The S/O/W type emulsion was slowly stirred with a conventionalpropeller agitator for 3 hours. After methylene chloride was vaporized,the resulting microcapsules were collected by centrifugation, washedwith purified water and subjected to freeze drying.

[0182] To the obtained microcapsules 20 mg were added ethyl acetate 5 mland 0.1 M acetic acid buffer solution (PH=4) 1 ml. The mixture wasshaken for 10 minutes and centrifuged at 2, 500 rpm for 10 minutes.Ultraviolet (UV) absorption (294 nm) of the resulting ethyl acetatelayer was measured with an spectrophotometer (Beckman DU 7400) todetermine the content of vitamin E in the microcapsules. The content ofvitamin E in the microcapsules was 10%.

Working Example 6

[0183] In a mixture of methylene chloride 2.0 ml and acetonitrile 0.4 mlwere dissolved lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50 (mole %), weight-average molecular weight: 15,000) 1350 mgand vitamin E 300 mg to give an oil phase. In the oil phase wasdispersed hGH/Zn complex 75 mg obtained in Reference Example 1. Thedispersion was subjected to ultrasonication for 5 minutes and then to asmall homogenizer (Polytron) at 15,000 rpm for 1 minute to pulverize itto microparticles. According to a similar method described in WorkingExample 5 the S/O dispersion was prepared, and thereafter the S/O/W typeemulsion was prepared, and finally freeze dried microcapsules wasprepared. In the prepared microcapsules, the content of vitamin E was17% when determined by a method described in Working Example 5.

Comparative Example 2

[0184] In a mixture of methylene chloride 2.0 ml and acetonitrile 0.4 mlwas dissolved lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50 (mole %), weight-average molecular weight: 15,000) 1425 mg togive an oil phase. In the oil phase was dispersed Met-hGH/Zn complex 75mg obtained in Reference Example 1. The dispersion was subjected toultra-sonication for 5 minutes and to a small homogenizer (Polytron) at15,000 rpm for 1 minute to pulverize it to microparticles. According toa similar method described in Working Example 6 the S/O dispersion wasprepared, and thereafter the S/O/W type emulsion was prepared, andfinally freeze dried microcapsules was prepared.

Test Example 2

[0185] The microcapsules (15 mg/kg as hGH content) obtained in the aboveWorking Example 5, Working Example 6 and Comparative Example 2 weresubcutaneously administered to SD rats (male, 6 weeks) and the serumlevel of hGH was determined by RIA, respectively. The thus obtainedresults were shown in FIG. 2. In FIG. 2, the curve A shows the serumlevel of hGH administered by the microcapsules of Comparative Example 2and the curves B and C show the serum level of the drug administered bythe microcapsules of Working Examples 5 and 6, respectively. The plasmalevel of hGH 1 hour after administration of the microcapsules ofComparative Example 2, Working Example 5 and Working Example 6 wererespectively 813 ng/ml, 633 ng/ml and 844 ng/ml. Bioavailabilities (BA)calculated from AUC (area under curve) of drug blood level-time until 2weeks after administration were respectively 42%, 56% and 57%, comparedwith intravenous administration of hGH solution. Initial burst (IB)which was calculated by the ratio of AUC until 1 day afteradministration relative to AUC until 2 weeks after administration wererespectively 83%, 79% and 72%.

[0186] In the microcapsules containing 10% vitamin E in an oil phaseprepared according to Working Example 5, increase of the plasma level(C1h) of hGH at an early stage (1 hour) after administration wassuppressed, the high serum plasma level of hGH was maintained for 2weeks, and increase of BA and suppress of IB were succeeded.

[0187] In the microcapsules containing 17% vitamin E in an oil phaseprepared according to Working Example 6, no remarkable change in C1h wasobserved, the high serum plasma level of hGH was maintained for 2 weeks,and increase of BA and suppress of IB were succeeded.

Working Example 7

[0188] In distilled water 1 ml were dissolved Compound A dihydrochloride500 mg and L-arginine 300 mg to give an inner aqueous phase. Inmethylene chloride 7.5 ml were dissolved lactic acid/glycolic acidcopolymer (lactic acid/glycolic acid=50/50 (mole %), weight-averagemolecular weight 8,000) 3950 mg and vitamin E 250 mg to give an oilphase. The oil phase was added to the inner aqueous phase, and themixture was emulsified with small homogenizer (Polytron) to give a W/Otype emulsion. The W/O emulsion was emulsified in 0.1% PVA solution 800ml (an outer aqueous phase) containing 2.7% NaCl which was cooled to 15°C. with using homomixer to give a W/O/W type emulsion. Then, the W/O/Wtype emulsion was slowly stirred with a conventional propeller agitatorfor 3 hours. After hardening of the microcapsules with evaporation ofmethylene chloride, the microcapsules were collected by centrifugation.The collected microcapsule were washed with purified water and subjectedto freeze drying. In the microcapsule, entrapment ratio of the drug was90%.

Working Example 8

[0189] In distilled water 1 ml were dissolved Compound A dihydrochloride500 mg and L-arginine 300 mg to give an inner aqueous phase. Inmethylene chloride 15 ml were dissolved lactic acid/glycolic acidcopolymer (lactic acid/glycolic acid=50/50 (mole %), weight-averagemolecular weight 10,000) 3160 mg, lactic acid/glycolic acid copolymer(lactic acid/glycolic acid=50/50 (mole %), weight-average molecularweight 7,000) 790 mg and vitamin E 250 mg to give an oil phase. The oilphase was added to the inner aqueous phase, and the mixture wasemulsified with small homogenizer (Polytron) to give a W/O typeemulsion. The W/O emulsion was emulsified in 0.1% PVA solution 800 ml(an outer aqueous phase) containing 2.7% NaCl which was cooled to 15° C.with using homomixer to give a W/O/W type emulsion. Then, the W/O/W typeemulsion was slowly stirred with a conventional propeller agitator for 3hours. After hardening of the microcapsules with evaporation ofmethylene chloride, the microcapsules were collected by centrifugation.In the microcapsule, entrapment ratio of the drug was 91%.

EFFECT OF THE INVENTION

[0190] According to the present invention, the microcapsules whichcontains high amount of a physiologically active substance and stablerelease of the drug with less initial release can be prepared by addinga fat and oil which is dissolved in an organic solvent containingbiodegradable polymer without causing phase separation. By using saidmicrocapsules, side effect of the physiologically active substance canbe decreased, the physiologically active substance can be administeredfor a long time, and compliance of patients can be improved due todecrease of the number of administrations.

What is claimed is:
 1. A process for producing a sustained-releasemicrocapsule of a water-soluble physiologically active substance, whichcomprises forming a w/o type emulsion comprising a solution containing awater-soluble physiologically active substance as an inner aqueous phaseand an uniform organic solvent solution containing a biodegradablepolymer and a fat and oil as an oil phase, and removing the organicsolvent.
 2. A process according to claim 1, wherein the w/o typeemulsion is dispersed in an aqueous phase, and the organic solvent isremoved by in-water drying.
 3. A process according to claim 1, whereinthe inner aqueous phase is a solution containing a water-solublephysiologically active substance and a basic substance.
 4. A processaccording to claim 1, wherein the water-soluble physiologically activesubstance is a polypeptide the molecular weight of which ranges fromabout 200 to about 80,000.
 5. A process according to claim 1, whereinthe water-soluble physiologically active substance is an integrinantagonist.
 6. A process according to claim 5, wherein the integrinantagonist is a GPIIb/IIIa antagonist.
 7. A process according to claim6, wherein the GPIIb/IIIa antagonist is a 2-piperazinone-1-acetic acidderivative represented by the formula (I):

wherein A¹ and A² are independently a proton-accepting group or a groupconvertible into a proton-accepting group, D is a spacer having a 2- to6-atomic chain optionally bonded through a hetero atom and/or a 5- or6-membered ring (provided that the 5- or 6-membered ring is counted as2- or 3-atomic chain, depending on its bonding position), R¹ is ahydrogen atom or hydrocarbon group, R² is a hydrogen atom or a residualgroup formed by removing —CH(NH₂)COOH from an α-amino acid, or R¹ andR²may be combined to form a 5- or 6-membered ring, P is a spacer havinga 1- to 10-atomic chain optionally bonded through a hetero atom and/or a5- or 6-membered ring (provided that the 5- or 6-membered ring iscounted as 2- or 3-atomic chain, depending on its bonding position), Yis an optionally esterified or amidated carboxyl group, and n is aninteger of 0-8; or a salt thereof.
 8. A process according to claim 7,wherein the 2-piperazinone-1-acetic acid derivative (I) is(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid or a salt thereof.
 9. A process according to claim 7, wherein the2-piperazinone-1-acetic acid derivative (I) is(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid hydrochloride.
 10. A process according to claim 7, wherein the2-piperazinone-1-acetic acid derivative (I) is(S)-4-(4-guanidinobenzoylamino)acetyl-3-[3-(4-guanidinobenzoylamino)]propyl-2-oxopiperazine-1-aceticacid dihydrochloride.
 11. A process according to claim 1, wherein thebiodegradable polymer is an aliphatic polyester.
 12. A process accordingto claim 11, wherein the aliphatic polyester is a lactic acid/glycolicacid copolymer.
 13. A process according to claim 1, wherein the fat andoil is an oil soluble vitamin.
 14. A process according to claim 13,wherein the oil soluble vitamin is α-tocopherol.
 15. A process accordingto claim 1, wherein the final concentration of the fat and oil in awhole content of the sustained-release microcapsule is about 3% to about30% (w/w).
 16. A process according to claim 1, wherein the W/O typeemulsion comprising a solution containing a water-solublephysiologically active substance and a basic substance as an inneraqueous phase and an uniform organic solvent solution containing abiodegradable polymer and a fat and oil as an oil phase is dispersed inan aqueous phase to form a W/O/W type emulsion, and the organic solventis removed by in-water drying.
 17. A process according to claim 3 or 16,wherein the basic substance is a basic amino acid.
 18. A processaccording to claim 17, wherein the basic amino acid is L-arginine.
 19. Aprocess according to claim 3 or 16, wherein the final concentration ofthe basic substance in a whole content of the sustained-releasemicrocapsule is about 1% to about 8% (w/w).
 20. A process for producinga sustained-release microcapsule, which comprises removing an organicsolvent from a S/O type dispersion where a metal complex of awater-soluble physiologically active peptide is dispersed in an uniformorganic solvent solution containing a biodegradable polymer and a fatand oil.
 21. A process according to claim 20, wherein the S/O typedispersion is dispersed in an aqueous phase to form a S/O/W typeemulsion, and the organic solvent is removed by in-water drying.
 22. Aprocess according to claim 20, wherein the water-soluble physiologicallyactive peptide is human growth hormone.
 23. A process according to claim20, wherein the metal complex of the water-soluble physiologicallyactive peptide is a zinc complex of human growth hormone.
 24. Asustained-release microcapsule produced by the process according toclaim
 1. 25. A sustained-release microcapsule produced by the processaccording to claim
 20. 26. Use of a fat and oil for the manufacture of asustained-release microcapsule of a water-soluble physiologically activesubstance, said microcapsule being produced by forming a w/o typeemulsion comprising a solution containing the water-solublephysiologically active substance as an inner aqueous phase and anorganic solvent solution containing a biodegradable polymer as an oilphase, and removing the organic solvent.
 27. Use of a fat and oil forthe manufacture of a sustained-release microcapsule of a metal complexof a water-soluble physiologically active peptide.